Cellular adhesion to the extracellular matrix involves members of the integrin family of cell adhesion receptors. Recent evidence indicates that integrins not only serve as receptors for cell adhesion but also directly transduce signals that regulate cellular proliferation, differentiation, gene expression, cell morphology and migration. Therefore the goals of this proposal will address the structural basis of integrin-ligand recognition as it relates to signal transduction events, integrin- cytoskeletal association and the resulting effects on cellular morphology and migration. This proposal will focus on the post-ligand binding events promoted by two homologous integrins, avbeta3 and avbeta5 that share the ability to bind vitronectin. We will take advantage of our recent findings that while both aV-containing integrins contribute to cell attachments to vitronectin only avbeta3 localizes to focal contacts. Based on these findings and the primary sequence comparison of beta3 and beta5 it is apparent that the structurally unrelated cytoplasmic tails of these proteins may be responsible for distinct signal transduction events and differential association with the actin-cytoskeleton. This proposal will focus on four related goals. First, we will characterize the molecular events involved in the binding of vitronectin by avbeta3 based on our recent evidence that this occurs in two steps: 1) RGD recognition, 2) followed by a molecular stabilization of this interaction that appears to be RGD-independent. This will include an examination of the regions in the ligand vitronectin to which av integrins bind using vitronectin fusion proteins, peptide, and anti-vitronectin monoclonal antibodies that block cell adhesion. Secondly, we will determine why integrin avbeta3 and avbeta5 differ in their ability to associate with the actin cytoskeleton. We will focus on the cytoplasmic tails of beta3 and beta5 in these studies. Specifically, mutant and chimeric integrins (beta3/beta5) with altered cytoplasmic tails will be transiently and/or stably expressed to allow us to investigate the role of the integrin beta subunit cytoplasmic domain in this process. A third goal of the proposed studies will be to examine the ability of integrins avbeta3 and avbeta5 to transmit early signals and whether the structural difference in these integrins leads to distinct cell signals. Initially we will measure the ability of these receptors to regulate the Na-H antiporter, intracellular calcium, inositol lipid synthesis and tyrosine phosphorylation. Finally, we will determine the role of these integrins in the post-ligand binding events of cell spreading, migration and proliferation. The objectives of this proposal are therefore designed to gain an understanding at the molecular level of how integrin occupancy mediates cellular responses to the extracellular matrix and whether these events facilitate the progression or metastasis of human tumor cells.